A. Field of the Invention
The present invention relates in general to a method and to a kit of materials for performing T cell repertoire analysis and clinical applications therefore, and more particularly to a method and a kit of materials for determining intrafamily gene fragment length profiles of the T cell receptor .alpha. and .beta. chain CDR3 regions.
B. Background
The T lymphocytes are the primary mediators of cellular immunity in humans, occupying an essential role in immune responses to infectious agents (e.g., viruses and bacteria) and in the body's natural defenses against neoplastic diseases. Likewise, T lymphocytes play a central role in acute graft-versus host disease, wherein the immune system of a host attacks (rejects) implanted tissue from a foreign host, in autoimmune disorders, in hypersensitivity, in degenerative nervous system diseases, and many other conditions. A T cell immune response is characterized by one (or more) particular T cell(s) recognizing a particular antigen, secreting growth-promoting cytokines, and undergoing a monoclonal (or oligoclonal) expansion to provide additional T cells to recognize and eliminate the foreign antigen.
Each T cell and its progeny are unique by virtue of a structurally unique T cell receptor (TCR) expressed thereby, which recognizes a complimetary, structurally unique antigen. In the vast majority of T cells (.alpha..beta. T cells), the T cell receptor is a heterodimer comprised of an alpha- (.alpha.-) and beta- (.beta.-) polypeptide chain covalently linked to each other by disulfide bonds. Alpha- and beta-chains both are comprised of an amino-terminal variable (V) region joined to a constant (C) region by an intermediate joining (J) region (and in the case of the .beta.-chain, by a diverstity (D) region as well). The diversity of TCRs is thought to be as large as that of antibodies, arising from the many different combinations of V.alpha., J.alpha., C.alpha. gene segments and V.beta., J.beta., D.beta., and C.beta. gene segments produced by genetic recombination events.
Within the T cell receptor .alpha. and .beta. chain variable regions are hypervariable regions similar to those found in immunoglobulins, where they form the principal points of contact with antigen and thus are referred to as complementarity determining regions (CDR). Based on the analogy with immunoglobulins, these TCR hypervariable regions are thought to loop out from connecting .beta.-sheet TCR framework sequences. Two CDRs (CDR1 and CDR2) are postulated to contact predominantly major histocompatability complex (MHC) peptide sequences, whereas a third, centrally-located CDR (CDR3) is believed to contact antigen bound in a groove between 2 MHC .alpha.-helical) peptides. See Bjorkman et al., Nature, 329: 512 (1987); Chothia et al., EMBO J., 7: 3745 (1988); Davis et al., Nature, 334: 395 (1988); and Brown et al., Nature, 364: 33 (1993). Since both .alpha. and .beta. chain CDR3 lengths are consistent with directly contacting peptide antigens, they have been postulated to be constrained in size because of their evolutionary selection for binding to peptide-MHC complexes. Rock et al., J. Exp. Med., 179: 323 (1994). Maximum sequence diversity of the TCR .beta. chain is reported to be in the CDR3 region at amino acid positions 96 through 105. Kabat et al., Sequences of Proteins of Immunologic Interest, Fifth edition, U.S. Dept. of Health and Human Services, NIH publication 91,3242 (1991). This diversity presumably is used in antigen recognition.
In addition to sequence diversity, TCR .alpha. and .beta. chains exhibit length diversity in their CDR3 regions due to nucleotide deletions and additions which occur during genetic recombination. Davis et al., (1988). Sequence data has shown the median CDR3 length in humans for both the .alpha. and .beta. chain to be 9 amino acids (a.a.) with a range of 6-12 a.a. Rock et al., (1994). Qualitative studies have suggested a 5-16 a.a. range of CDR3 lengths. Hingorani et al., J. Immunol., 151: 5762 (1993); Gorski et al., J. Immunol., 152: 5109 (1994).
Because of the integral role of T lymphocytes in the immune response, the analysis of the T cell repertoires involved in local and systemic immune responses is beginning to play an important role in many clinical situations, including auto-immunity, response to infectious antigens, alloimmunity, and tumor immunity. Gorski et al., (1994). Intensive investigative efforts have been directed to developing improved methods for monitoring the T cell repertoire to better understand, monitor, and modulate the immune system. Many of the more successful advances in T cell repertoire analysis have involved polymerase chain reaction (PCR) methodologies directed to measuring T cell receptor repertoires. See generally Cottrez et al., J. Immunol. Methods, 172: 85-94 (1994).
For example, Oaks et al., Am. J. Med. Sci., 309(1): 26-34 (1995) reported a PCR-based method of T cell repertoire analysis comprising extracting RNA from a cell sample, synthesizing cDNA from the RNA, and amplifying aliquots of the cDNA via PCR (around 40 cycles) using family-specific V.alpha. and V.beta. oligonucleotide primers. The PCR products were analyzed by electrophoresis on a 2% agarose gel followed by Southern blotting using .alpha.-chain or .beta.-chain constant region gene probes, wherein expression of a specific TCR V.alpha. or V.beta. family was considered positive if a distinct band was detected. The method was useful for distinguishing tissue rejection lesions versus non-rejection lesions in cardiac allograft patients. However, the Southern blot analysis provides suboptimal information about the T cell repertoire within a particular V.alpha. or V.beta. gene family. See also Dietrich et al., Blood, 80(9): 2419-24 (1992).
In European Patent Application No. 0 653 493 A1, filed Apr. 30, 1993, the inventors reported a PCR-based method of T cell repertoire analysis comprising extracting RNA from a cell sample, synthesizing cDNA from the RNA, and amplifying aliquots of the cDNA via PCR using family-specific V.beta. oligonucleotide primers. The PCR products were then analyzed using a "single strand conformation polymorphism" (SSCP) technique wherein the PCR-amplified cDNA is separated into single strands and electrophoresed on a non-denaturing (urea-free) polyacrylamide gel, whereby DNA fragments having the same length are made further seprarable by differences in "higher order structure." Using this method, the amplified DNA from peripheral blood lymphocytes reportedly is observed generally as a "smear" whereas the detection of a single band amidst a smear is indicative of a T cell clonal expansion.
Cottrez et al., supra, reported a PCR-based method of T cell repertoire analysis comprising extracting RNA from a cell sample, synthesizing cDNA from the RNA using oligo-dT primers, and amplifying aliquots of the cDNA via PCR (around 25 cycles) using family-specific V.beta. oligonucleotide primers. The PCR products were analyzed on a DNA sequencer and reportedly contained 6-11 discrete fragment peaks spaced by 3 base pairs in length, representing "all" various sizes of the CDR3 region. See also Gorski et al., J. Immunol., 152: 5109-5119 (1994).
Puisieux et al., J. Immunol., 153: 2807-18 (1994), reported a PCR-based method of T cell repertoire analysis comprising determining VDJ junction size patterns in twenty-four human TCR V.beta. subfamilies. The TCR V.alpha. subfamilies were not characterized. These investigators employed the method to analyze T cells infiltrating sequential malignant melanoma biopsies for the presence of clonal expansions, and detected such expansions over a more or less complex polyclonal background. Their study highlights the utility of T cell repertoire analysis methods for monitoring neoplastic conditions and treatments for such conditions.
The method of T cell repertoire analysis of Puisieux et al. reportedly includes the steps of extracting RNA from cells, synthesizing cDNA from the RNA using oligo-(dT) primers, and amplifying aliquots of the cDNA via PCR using family-specific V.beta. oligonucleotide primers. Potential clonal expansions in the PCR products were tentatively identified in families where a single fluorescence peak (on a sequencing gel) corresponded to 40% of the total fluorescence intensity of all of the peaks in the family. To "refine" the T cell repertoire analysis, a second set of V.beta. family-specific PCR reactions were conducted and then aliquots from each of the PCR reactions of interest were further subjected to primer extension "run off" reactions using a fluorophore labelled C.beta. primer and/or using thirteen J.beta.-family-specific, fluorophore-labelled J.beta. primers. The run-off reaction products were then analyzed on additional sequencing gels.
The same investigative group has more recently elaborated on their T cell repertoire analysis methods. See Pannetier et al., Immunol. Today, 16: 176-181 (1995). The group reports that the V.beta. families are easier to analyze by PCR than V.alpha. families. Nonetheless, their V.beta. analysis methods involve twenty-five V.beta. family-specific PCR amplifications (each of which yields an average of eight peaks), twenty-five C.beta. "run-off" reactions, and 325 J.beta. "run-off" reactions (25 V.beta..times.13 J.beta.=325). Each "run-off" reaction is analyzed by electrophoresing an aliquot on a polyacrylamide gel. See also Cochet et al., Eur. J. Immunol., 22: 2639-2647 (1992) (wherein the same investigative group reports the earlier use of a similar method to analyze the T cell repertoire in mice); and Dietrich et al., Blood, 84(8): 2815-20 (1994) (demonstrating the utility of T cell repertoire analysis methods for monitoring tissue transplant patients for graft-versus-host disease). A long felt need exists for more rapid methods of T cell repertoire analysis, e.g., methods wherein fewer PCR reactions and electrophoresis reactions are required, wherein putative peaks representing clonal expansion events are more rapidly identified, and wherein the need to optimize interfamily PCR conditions is reduced or eliminated. Moreover, a long felt need exists for methods of T cell repertoire analysis which provide an analysis of TCR V.alpha. chains. Preferably, the more rapid methods provide sensitivity greater than or equal to existing methods in their ability to detect clonal expansion events, including clonal expansions of T cells whose T cell receptor comprises an alpha-chain and/or beta-chain having a low-prevalence CDR3 length.